Early in vertebrate development, mesodermal cells become committed to the cardiogenic lineage. Soon thereafter, these committed precursors begin to differentiate into cardiac myocytes. The molecular basis of cardiac differentiation is not yet understood. Our new data demonstrate, in molecular terms, the embryonic period when committed cardiac progenitor cells are converted to initially differentiated cardiac myocytes. Using an antiserum against the conserved second helix of the myogenic helix-loop-helix (HLH), we provide immnochemical evidence that a HLH protein(s) [called H2] is specifically expressed during the period when committed cardiac progenitors are converted to differentiated myocytes (stage 7-15 of avian development). Western blot and immunoprecipitation analyses demonstrate the presence of a 5OKd protein which is specifically expressed at this time which is reactive with this antibody. Electrophoretic mobility shift assays (EMSA) show the stage specific expression of a DNA binding proteins interactive with muscle specific enhancer sequences and that this DNA/protein complex specifically reacts with the anti-second helix antibody. In a second set of experiments using polymerase chain reaction (PCR) analysis we demonstrate the presence of an additional HLH protein (called Atwist), which is homologous to Drosophila and Xenopus twist and is expressed throughout development. Our aims in the proposed grant are clone and characterize these regulatory proteins specifically expressed in the heart using cDNA cloning techniques. Taken together, these studies should provide an understanding of the cellular and molecular basis of cardiac myogenesis.